The recent identification of the cystic fibrosis (CF) gene, the cystic fibrosis transmembrane conductance regulator (CFTR) gene (Riordan JR et al., Science 1989;245:1066-73), revealed that the heme belongs to a family of membrane transport molecules that includes the multi-drug resistance genes. Approximately 70% of CF chromosomes contain a deletion of 3-base pairs resulting in the loss of a phenylalanine codon at amino acid position 508 (delta-F 508). The focus of this project is to identify new mutations in this gene that comprise the remaining 30% of CFTR gene mutations. Using the polymerase chain reaction technique, the coding regions of the gene have been examined from patients that have at least one chromosome without the common CF mutation (delta-F508). This year we report the identification of five additional mutations in the CF gene (in addition to the eight reported last year). Most of the mutations were initially identified using an assay for single- stranded conformation polymorphisms. All mutations were subsequently characterized by direct sequencing of the amplified DNA and can be assayed by restriction enzyme digestion or allele- specific oligonucleotide hybridization. The mutations fall into two classes: (1) insertions or deletions that introduce termination codons into the gene and are predicted to result in severely truncated protein products, and (2) point mutations in the putative membrane-spanning domains that replace charged amino acids with non-polar residues. In addition, we have cloned a region of the mouse CF gene homolog. Restriction mapping and Southern blotting experiments have indicated that this clone contains an exon that is highly homologous to exon 7 of the human CF gene. This exon was subcloned for use in homologous recombination/gene targeting experiments aimed at creating a disease model for CF.